WATER MICROBIOLOGY AND FERMENTATION Flashcards
Essential to all organisms
water
WATER-BORNE DISEASES
Bacterial
● Leptospirosis (Leptospira)
● Typhoid (S. enterica serotype typhi)
● Paratyphoid (S. enterica serotype paratyphi)
● Cholera (Vibrio cholerae)
● Bacillary Dysentery (E. coli, Shigella, Campylobacter, Salmonella)
WATER-BORNE DISEASES
Viral
● Viral Hepatitis (Hepatitis virus A and E)
● Poliomyelitis (poliovirus)
● Infant diarrhea (Rotavirus)
● Gastroenteritis and Traveler’s Diarrhea due to Norovirus
WATER-BORNE DISEASES
Protozoal and Parasitic
● Amoebiasis (Entamoeba histolytica)
● Giardiasis (Giardia lamblia)
● Schistosomiasis (Schistosoma)
● Roundworms, Whipworms (helminths)
microorganisms whose presence also indicates the probable presence of pathogenic microorganisms.
Indicator organisms
Example of indicator organisms (particularly used in water microbiology assessments) are
coliforms
Indicator of fecal/sewage contamination in food or water, thus indicating possible presence pathogenic bacteria.
coliforms
coliform characteristics
Rod-shaped (bacilli)
facultative anaerobe
non-spore forming
lactose-fermenting
gram-negative bacteria
why do we use indicator organisms and not tets the pathogen directly?
Indicator organisms are used instead of directly testing for pathogens because it’s impractical to test for every pathogen in every water sample
the main pathogen is also very small in numbers
agar used in water quality tests to distinguish coliforms and fecal coliforms that signal possible pathogenic microorganism contamination in water samples
inihibts the growth of most gram-positive bacteria
differentiates bacteria that ferment lactose
Eosin-Methylene Blue (EMB) Agar
● Based on statistical estimation using Most Probable Number of MPN
● MPN estimates the concentration of viable mcgs in a sample
● Sub-samples are inoculated in a series of tubes
Multiple Tube Fermentation Technique
(MTFT)
Multiple Tube Fermentation Technique
(MTFT) Typically composed of 3 stages
presumptive, confirmed, and
completed
Multiple Tube Fermentation Technique
(MTFT)
Stage 1: Presumptive test in Lactose Broth with Durham tube
After incubation: Check for yellowing/gas production in the tubes is (+) for presumptive test
Stage 2: Confirmed Test using EMBA
After incubation: Check for dark colored colonies or green metallic sheen colonies is (+) for confirmed test
Stage 3: Completed Test via IMViC and Microscopy
After Gram Stain: Look for gram negative, non-spore forming rods is positive for Completed Test
After IMViC: Indole (+), Methyl Red (+), Voges Proskauer (-), Citrate (-) is positive for E. coli
Test if tryptophan is converted to indole
Indole Test
Indole Test
Indicator
mechanism
positive, negative result
(+) Red ring is observed when Kovac’s reagent is reacts with indole.
Detects lower pH due to fermentation of glucose
Methyl Red
Methyl Red
Indicator
mechanism
positive, negative result
Methyl red indicator
Yellow to red if acidic products are produced.
● Test if mcg uses citrate
Citrate Utilization Test
Citrate Utilization Test
Indicator
mechanism
positive, negative result
● Citrate permease
● If mcg uses citrate NH4 salts are converted to NH3 which rises the pH
● Bromothymol blue (green to blue)
● To check acetoin production from glucose fermentation
Voges Proskauer
Voges Proskauer
Indicator
mechanism
positive, negative result
● In the presence of oxygen, alkali and naphthol, acetoin is converted to diacetyl w/c condenses with guanidine from peptone w/c produces a pinkish polymer
IMViC test stands for
Indole Test
Methyl Red
Voges Proskauer
Citrate Utilization Test
a statistical method used to estimate the viable numbers of bacteria in a sample by inoculating broth in 10-fold dilutions
MTFT results are interpreted using this Table
Most Probable Number (MPN) Table
● Determine microbial load of sample by calculating colony forming units per unit of the sample (cfu/g or cfu/ml)
● Done using Plate Count Agar (PCA)
● Good quality water should have <100 cfu/mL
● HPC indicates efficiency of water treatment (chlorination, filtration, sedimentation etc.)
HETEROTROPHIC PLATE COUNT
Series of sequential dilution used to reduce dense cultures to obtain usable (countable) concentration
This is performed in conjunction with plating e.g., spread or pour plating to estimate the microbial load in a sample
Serial Dilution
calculate colony forming units per unit of sample:
Cfu/ml = (no. of colonies*/amount inoculum plated) x dilution factor
if done in duplicates, use average no. of colonies
colonies are the usable counts
30-300
<30 colonies
TFTC
> 300 colonies
TNTC
Inoculum plated spread plate
0.1 mL
Inoculum plated pour plate
1.0 mL
count the number of colonies of microorganisms that have grown on an agar plate prepared from a sample
quebec colony counter
Metabolic pathway of oxidation-reduction to
produce energy.
Performed by heterotrophic organisms
anaerobically (in absence of oxygen).
In this process, there is no net oxidation of
the fuel.
FERMENTATION
FERMENTATION Common fuel
sugars, fatty acids, amino acids, purines and pyrimidines.
types of fermentation
Lactic Acid or Homolactic Fermentation
Ethanol or Alcohol Fermentation
one molecule of glucose is ultimately converted to two molecules of lactic acid.
Lactic Acid or Homolactic Fermentation
one molecule of glucose yields carbon dioxide and ethanol in addition to lactic acid
Heterolactic fermentation
WINE MAKING
Preparation of Fruit Mash
1.0 kg fruit
Osterize and liquify in a blender in 250 ml of water
Fill the fruit mash with water up to 2 Liters in a large beaker
Add 500 g sugar and stir to dissolve
Test pH and adjust it to 4.0-4.7. For highly acidic mash, adjust using CaCO3
Heat at 65 °C for 30 minutes (pasteurize). Cool and transfer to a demijohn gallon
WINE MAKING
Fermentation Proper
Add a teaspoon yeast (Saccharomyces cerevisiae)
Set-up the demijohn and the limewater trap accordingly
Ferment for 1 week and or until no bubbles are seen in the limewater trap
WINE MAKING
Wine Harvesting
After fermentation, filter the mash through a cheesecloth
Add one beaten egg in the filtrate
Pasteurize at 65 °C for 30 minutes the mixture then decant in sterile bottles.
are large vessels for fermenting wine, cider and mead. They are often made from thick glass
demijohn gallon
optimum ph for wine mash
4.0-4.7
trap for wine demijohn, to determine whether carbon dioxide was produced. When this reacts with CO2 it becomes milky.
Limewater Trap
Ca (OH)2
is a sour milk product attributed to the presence of lactic acid produced by bacteria that underwent fermentation
Yoghurt
Commonly used bacteria in yogurt production:
● Streptococcus thermophilus
● Lactobacillus bulgaricus
YOGURT MAKING
Put the 1L of Fresh milk in large beaker
Add Skim milk powder then stir
Heat the mixture in a water bath at 90 °C for 10 minutes
Chill the mixture in an ice bath
Add the starter culture
Incubate at 43-46 °C for at least 4 hours or until clotting is observed.
brings the acidity to the desired level.
Tartaric, citric or malic acid
neutralize excess acidity
Potassium tartrate or calcium carbonate
enzymes that clears the wine
Tarnin and pectolytic
produces fizz or effervescence (from secondary fermentation)
Invert or cane sugar
arrests the fermentation in the making of appetizer and dessert wines.
Brandy
wines that contains low sugar
Dry table wines
Are found in table and sparkling wines
EtOH (18.5%)
found at 10-175 mg/100mL of wines.
Higher alcohols e.g., isoamyl and butyl-OH
Acids such as ________ are also produced but in excess this can lead to spoilage of wine.
formic, acetic and propionic
Table wines typically contain 0.5 to 1.0%
titratable acidity
most desirable level of titratable acidity
0.65%
Volatile fatty acids (VFAs) are produced through heterolactic fermentation
true
Volatile fatty acids (VFAs)
acetic, propionic, and butyric acids
Enzyme and reagent in indole test
tryptophanase enzyme
Kovac reagent
